The present invention relates to an improved assay method for the immunological determination of intact, either classical or .alpha.1-homotrimer, aminoterminal propeptide of type I procollagen in a sample such as serum, and to preparation of an antiserum suitable for use in this method.
Type I collagen is the most abundant collagen type in the human body. Most of it is found in the bones, although other soft connective tissues also contain considerable amounts of it. In mineralized tissues such as bone, about 90% of the organic matrix is type I collagen, there being no other collagen types in these tissues. Thus analysis of type I collagen is important in diseases affecting bones, such as metabolic bone diseases including primary and secondary osteoporosis, bone metastases in breast or prostatic carcinomas, rheumatoid arthritis and various genetic diseases such as osteogenesis imperfecta. It is also known that the collagen synthesis rate in bone affected by various hormones, e.g. growth hormone, thyroxine, cortisol and estrogens.
Type I collagen is synthesized as a procollagen, containing propeptide extensions at both ends of the molecule. The rate of type I collagen synthesis can thus be assessed by determining the amount of propeptide liberated during the conversion of procollagen to collagen. It is known to assay the carboxyterminal propeptide of type I procollagen (abbreviated PICP) in serum (Melkko J, Niemi S, Risteli L, Risteli J: Clin. Chem. 1990; 36; 1328-1332). However, there are certain individuals in whom the elimination rate of this propeptide from the circulation is deteriorated, leading to very high serum levels of PICP, which are not related to the rate of synthesis of type I procollagen. On the other hand, the previous assays developed for the aminoterminal propeptide of type I procollagen have been based either on synthetic monomeric linear peptides (Ebeling P. R., Peterson J. M., Riggs B. L.; J. Bone Miner. Res. 1992; 7; 1243-1250 and Linkhart S. G., Linkhart T. A., Taylor A. D., Wergedahl J. E., Bettica P, Baylink D. J.: Clin. Chem. 1993; 39; 2254-2258) or on propeptides isolated from amniotic fluid (Teisner B, Boje Rasmussen H, Hoejrup P, Yde-Anderson E, Skjoedt K: APMIS 1992; 100; 1106-1114 and Price K. M., Silman R, Armstrong P, Grudzinskas J G: Clin. Chim Acta 1994; 224; 95-102) or cell culture medium (Jukkola A, Risteli L, Melkko J. Risteli J: J. Bone Miner. Res. 1993; 8; 651-657). Such assays have been found to detect two antigenic forms in human serum, are of different sizes. One of the forms corresponds to the authentic trimeric propeptide in size and the other is its further degradation product, which resembles the globular, so called Col1 domain of the propeptide.
The evidence available suggests that these small molecular weight degradation products are not derived from the further degradation of the liberated propeptide but most likely from the degradation of type I procollagen molecules that have retained the aminoterminal propeptide in the tissues (so called pN type I collagen). Such molecules are found on the surface of type I collagen fibers, mainly in soft connective tissues and temporarily in newly synthesised non-mineralized osteoid. Further, it has been shown that the authentic aminoterminal propeptide is eliminated from the circulation via the scavenger-receptor of the liver endothelial cells, whereas the smaller Col1 related degradation products are eliminated via the kidneys. Thus kidney disease, while decreasing the clearance of the Col1-related peptide, would greatly affect the serum concentration of such peptides and give false results with respect to collagen synthesis. Since the intact aminoterminal propeptides of type I procollagen are not excreted into the urine, the assay of the present invention is applicable to serum and other biological fluids except urine. In addition, the catabolic status often encountered in seriously ill patients will increase the tissue degradation of pN type I collagen, similarly increasing the serum Col1 concentration and giving false information on the anabolic capacity of the patient.
It would, therefore, be desirable to solve the interpretation problem of the results caused by the different origins and elimination routes of the two propeptide forms and to provide a quantitative method, which is quick and simple to practise, for assaying only the intact aminoterminal propeptide of type I procollagen in human serum, to the exclusion of the Col 1-related degradation products.
The intact propeptide is in the form of a trimer, whereas the degradation product comprises the monomeric globular Col1 domain of the .alpha.1-chain of type I procollagen. When isolating the intact, trimeric aminoterminal propeptide of type I procollagen from pleural fluid from a carcinoma patient, two separate propeptides were surprisingly discovered, which differed with respect to their constituent chains. Two different polypeptide chains were found in the propeptide form eluting first from the DEAE-chromatography column, since the classical type I procollagen is a heterotrimer containing two pro .alpha.1-chains and one pro .alpha.2-chain of type I procollagen. The more acidic propeptide lacked the second polypeptide chain that moves faster in electrophoresis and is known to be derived from the pro .alpha.2-chain of type I procollagen. This atypical propeptide was derived from the .alpha.1-homotrimer type I procollagen containing three identical pro .alpha.1-chains. Such a collagen has been previously described in a fully processed form from the tissues of patients with certain disease states e.g. breast, lung and stomach carcinomas, but the corresponding propeptides have not been identified. Since the aminoterminal portion of the pro .alpha.2-chain is truncated with respect to the pro .alpha.1-chain and lacks the whole of the Col1 domain, the .alpha.1-homotrimer propeptide has three Col1 domains and the classical propeptide only two. Since the Col1 domains are phosphorylated, these two intact propeptides can be separated from each other on the basis of the difference in their charge, e.g. using DEAE-chromatography at pH 5.0. Since the .alpha.1-homotrimer propeptide has three Col1 domains and the classical propeptide only two, the latter elutes earlier in anion-exchange chromatography. Both these propeptides are susceptible to being denatured at low pH (less than 5.0); something which is not easily detected but can cause problems when producing antibodies if proper precautions are not taken.
The antisera used previously for assaying the aminoterminal propeptide of type I procollagen has affinity not only for the intact trimeric propeptide, but to a considerable extent also for the monomeric form. This is due to the nature of the antigen used to raise the antisera. When a linear synthetic peptide, or a propeptide derived from amniotic fluid and isolated in a monomeric form, is used for raising the antibodies, these antibodies react preferentially with the monomeric Col1 degradation products. On the other hand, when the propeptide is isolated from cell culture fluid, it is first purified in the form of a procollagen, which is later digested by bacterial collagenase to liberate the propeptide from the collagen proper. Since the authentic propeptide still contains a collagenous domain, part of the bacterial collagenase binds to it, but is not able to digest the propeptide during the short in vitro incubation and isolating procedures. Consequently, when such a propeptide is used as an immunogen, the enzyme is capable of functioning, leading to in vivo degradation of the propeptide and production of antibodies towards its degradation products. In addition, it is also necessary to avoid such conditions and methods (e.g. high performance liquid chromatography at pH lower than 5.0) which would denature even a small portion of the propeptide during the isolation procedure of the aminoterminal propeptide of type I procollagen. This is beacuse, in contrast to the corresponding propeptide of type III procollagen (EP-B-0-304292), the aminoterminal of type I procollagen has no interchain disulphide bonds, which would stabilize the structure and facilitate rapid renaturation after minor accidental denaturation during the purification procedure. Thus, removal of contaminating enzymes from type III procollagen can be effected by reverse phase separation at low pH. This would, however, lead to denaturation of trimeric PINP of type I procollagen because of its lack of stabilising disulphide bonds.